The object of the present invention is a system for measuring biochemical and medical samples, the said measuring being carried out by imaging. The objects of imaging are mainly regular macro-size sample matrices, gels, Petri dishes or completely free-form samples, such as, for example, biological sections. The signal to be imaged is ultraviolet light, visible region light, or infrared light.
Light-producing mechanisms are:
1) luminescence, such as chemiluminescence and bioluminescence, in which case the light p produced by each sample at different points of the sample is measured,
2) fluorescence, in which case the amount of emission light produced by special excitation light at different points of the sample is measured ed, and in addition
3) the amount of reflection, scattering, or absorption at different points of the sample, resulting from the illumination of the sample.
In known measuring devices many different types of sample plates are used, in which the number of wells may vary considerably. In a conventional sample plate there are, for example, 96 sample wells, in which case the amount of solution required for each well is 200 xcexcl. Another typical number of wells is 384 wells in a sample plate, in which case the amount of the solution required for each well is, for example, 200 xcexcl. Although these amounts are small as such, in cases where, for example, 100 000 samples are measured during one sample run, the overall costs are considerable. It obviously makes a marked difference whether 50 xcexcl of liquid or 1 xcexcl of the same liquid is used for one sample, which may, for example, be a single patient sample. The costs relating to the consumption of liquid are directly proportional to the volume used. In the course of measurements, during one set of sample measurements, that is, a sample run, considerable amounts, i.e. several litres of used solution is produced, the said solution often being hazardous waste. The residues of solution often contain radioactive and/or toxic chemical compounds. When numerous sample runs are performed daily (on parallel equipment and in different laboratories), the amount of toxic solution waste produced is considerable. Thus there are obvious reasons to reduce the amounts of solution significantly, that is, in practice to reduce the sample well volume.
To reduce the amount of liquid and to speed up measurements, sample plates with 864 wells are now being used in several measuring devices, in which case the amount of solution required is, for example, about 10 xcexcl. The aim has, however, been to reduce the size of the wells even further. There now already exist sample plates with 1536 wells in which the amount of solution required is now only 5-10 xcexcl, and possibly even as little as 1 xcexcl. In the near future the number of wells will increase furtherxe2x80x94sample plates with e.g. 9600 wells are being tested in laboratories.
Reducing the size of the sample wells has, however, caused problems, because a small-volume sample requires much better and more efficient measuring properties of the measuring device. Known devices do not usually meet these requirements without extremely complex constructions, or else their measuring times are unacceptably long, which affects the reliability of the measuring results and which also makes the use of parallel equipment compulsory in order to obtain a reasonable overall measuring time for the set of samples.
In luminescence and fluorescence measurements, the aim of reducing the volumes of the wells results in the amount of light from the sample well decreasing in proportion to the volume of the well. In luminescence measurements this means that either the measuring time must be extended correspondingly, or more sensitive measuring devices than the conventional ones have to be used. In fluorescence measurements the situation is different; the amount of fluorescent light produced is proportional to the efficiency, that is, intensity of the light of the excitation light. Especially when operating within the linear range of fluorescence, where the yield of emission light is directly proportional to the amount of excitation light, by doubling the intensity of the excitation light, for example, the amount of the emission signal obtained from the sample, that is, the amount of fluorescent light from the sample will also be double. In a measuring situation such as this it is obvious that the aim will be to increase the intensity of the excitation light considerably, so that shorter measuring times can be used.
In traditional fluorometry, one sample well is measured at a time. In such a case, the excitation light originating, for example, from a xenon flash lamp, is directed by means of focusing optics directly at the sample solution contained in one sample well. Each sample well is measured separately and in the same manner as the previous one.
In imaging, however, the situation is completely different. In this case, where the aim is to image all sample wells at the same time, the excitation light is directed at all the wells simultaneously. The easiest way to do this is by illuminating the entire sample plate with excitation light at one time. However, as the size of the sample plate may, for example, be 80 mmxc3x97120 mm, and the surface area of one well in a single plate comprising 1536 wells may be 1.5 mmxc3x971.5 mm, it is obvious that in order for the imaging to be successful, considerably more excitation light is required for imaging than for fluorometry, if the measuring times are to be of the same magnitude.
From this it follows that in fluorescence measurement it is difficult to obtain sufficiently powerful excitation light in the sample plate area, that is, in each sample well. It is also desirable that the uniformity of the excitation light field, that is, its intensity distribution over different parts of the sample plate should be as uniform as possible. The overall sensitivity of the measurement, that is, how small a specific part of a sample will be detected, is determined by that point in the excitation field which has the lowest intensity.
A continuous light source, for example, an arc lamp or a halogen lamp or any other device generating light continuously, is sufficient for prompt fluorescence. However, for time-resolved fluorescence a pulse mode light source is required, for example, a flash lamp or a pulsed laser. The length of the light pulse is of decisive importance for the sensitivity of the device, a property which in turn depends on the decay time of the fluorescence in the sample.
A pulse mode light source may be one of the following:
a) a flash lamp
b) a pulsed laser, such as the combination of an XeCl excimer laser and a dye laser or, for example, a nitrogen laser
c) a combination of a continuous light source and a light chopper; continuous lamps include an arc lamp, a halogen lamp, a continuous laser, and other lamps that produce light continuously.
For example, the light of an arc lamp is interrupted by means of a light chopper in the excitation/illumination path. In practice, the operation of this type of combination is rather ineffective, depending, however, on the application.
Time-resolved fluorescence is achieved by using a combination in which the light source is a pulse mode lamp and the camera acting as detector can be gated. The gating of the camera is a rapid shutter function. This is required because the illumination path leading to the camera must be shut at the moment when the light source flashes. It is only after this that the illumination path of the camera is opened. In practice, the gating of the camera can be done mainly by means of the following combinations of devices:
a) a sensitive camera, in front of which is a mechanical light chopper,
b) a sensitive camera, in front of which is a liquid crystal shutter device, which is triple if necessary.
c) an intensified charge coupled device camera
d) a gatable camera
In prompt fluorescence, it is possible to use a powerful lamp, because it applies spectral filtering. Excitation light and fluorescent light, which are at different wavelengths, can be separated from each other by means of a spectral filter. A disadvantage of prompt fluorescence is, however, that prompt fluorescence also easily comes from other fluorescent parts of the sample than from the fluorescent tracer being measured. This type of fluorescence at another point typically emits light over a wide wavelength region, which means that it is also emitted in the emission wavelength of the fluorescent tracer being measured. Since the optical filter in front of the camera has been selected according to the wavelength of the tracer, prompt fluorescent light originating from another single point than the tracer being measured can also enter the camera. In the image, these points may overlap, which sometimes makes it impossible to say, when analysing and observing the image, whether the signal comes from the fluorescent tracer being measured or from other interfering fluorescent light in the sample, which causes a background signal. Another disadvantage of prompt fluorescent imaging is that excitation light may also enter the camera, which causes more interfering background signals, which further reduces the measuring sensitivity of the measurement, that is, imaging.
In the imaging device relating to the invention, which does not apply time-resolved imaging as in prompt fluorescence and luminescence imaging (another embodiment), there is a dimmer in place of the chopper 13. In this case the optics also incorporate a mechanical shutter controlled electrically, pneumatically or otherwise, or a liquid crystal shutter device to shut the illumination path leading to the camera before the imaging signal is read electrically on the ccd matrix of the camera, which signal is formed into a digital image.
For imaging time-resolved fluorescence, a light chopper is needed in front of the detector, that is, in this case a sensitive, cooled ccd camera, by means of which the emission light path is interrupted for the short interval during which a short fluorescent excitation light pulse originating from a pulse mode light source is directed at the sample. After this the light chopper opens the illumination path leading to the camera to allow the passage of the long-living fluorescent light emitted from the sample to the camera. The chopper may, for example, be a mechanical rotating light chopper or a liquid crystal shutter device (LCD), which is most preferably located on the aperture plane of the imaging optics, which means that light chopping takes place in a controlled manner, without disturbing shadows or dark areas or other irregularities attributable lighting, being formed in the image.
If a separate light chopper is not used, time-resolved imaging can be performed by using an Intensified Charge Coupled Device (ICCD) camera.
It has been shown in practice that time-resolved (TR) fluorescence measurement is advantageous in many respects. If sufficiently short and powerful excitation light intensity is obtained for the sample in this measurement, there will also be sufficient time for measuring fluorescence after the excitation light has been switched off. However, for the above reasons, the reduced size of the sample wells and the increased number of wells have caused problems.
It is difficult to obtain uniform and sufficiently powerful excitation lighting over the entire sample plate area. At the same time, the exposure time should nevertheless be short enough to enable efficient measurement immediately after the excitation light has switched off. Long measuring times are unacceptable because in such a case the measuring device is too ineffective in practice.
Increasing the power of the excitation lamp has not provided a solution to this problem, because the pulse length of a powerful lamp is relatively long, for example, about 300 ps. The excitation fluorescence of many fluorescence measuring agents is halved already about 200 xcexcs after excitation, and thus the fluorescent light is covered under the excitation light.
Differences between cameras
1. Cooled CCD camera, that is, c-CCD (=cooled Charge Coupled Device): good resolution, wide dynamic range, good sensitivity, difficult to gate
2. Intensified Charge Coupled Device or ICCD camera: limited resolution, limited dynamics, good sensitivity, easy to gate.
The lens systems used in connection with excitation light sources also cause problems when the size of sample wells is reduced. Normally, the light emitted from the lens disperses so that the beams of light enter the sample well at an oblique angle, which means that the sample wells in the centre of the sample plate are obviously illuminated in a different manner than the sample wells at the edges of the sample plate.
The efficiency of measurement is also impaired by the fact that as the number of sample wells increases, the intermediate walls of the sample wells make up an increased relative proportion of the sample plate surface area. From this follows that the sample fluid in the sample well obtains too small a proportion of the excitation light. To achieve uniform and sufficiently intensive excitation lighting, the light beams must enter the sample well at a sufficiently small angle. According to one embodiment, a small entry angle of the excitation light beams is obtained by positioning the light source sufficiently far away from the sample.
Imaging lens systems
The question here is, therefore, of imaging at a fairly considerable downscaling-ratio, that is, about 1:5, because the size of the sample plate is about 80xc3x97120 mm and the size of the ccd matrix is, for example, 25xc3x9717 mm with current technology. It is easy to calculate the above-mentioned downscaling ratio on the basis of these figures. It would be more advantageous if the downscaling required for imaging were not so great, but closer, for example, to the ratio 1:2. However, ccd matrices are not widely available commercially in other than the above magnitude. It is, however, likely that the situation will change in the near future, which means that imaging will become more efficient from the point of view of measuring technique.
When imaging a sample, an image of the sample is formed on the camera on the basis of the emission light (luminescence, fluorescence, absorbancy) emitted by the sample. When using conventional optics such as a conventional objective camera lens, the parallax error of imaging is considerable due to the downscaling ratio used. In such a case the edges of the sample plate are imaged much less effectively than the sample wells in the centre of the plate. In order to be able to eliminate this drawback and to maximize the collection of light, a telecentric lens system should be used in imaging. In this case the sample wells in the centre and edges of the sample plate are imaged with equal efficiency.
According to a dictionary of optics, a telecentric lens is: xe2x80x9ca lens in which the aperture stop is located at the front focus, resulting in the chief rays being parallel to the optical axis in the image space, i.e. the exit pupil is at infinityxe2x80x9d (The Photonics Dictionary, 1993: Telecentric Lens).
For reasons relating to the basics of optical design, the lens system which collects light from the sample plate area must be designed so that at least two types of glass with different refractive indices are used. Moreover, good resolution is required of the optics, since the sample wells are imaged in small size on the camera. This means that the telecentric lens system has, for example, about 20 separate lens elements or even more.
From this it follows in turn that the total transmittance of the lens system is limited mainly to the visible area, that is, the wavelength 400-800 nm. In order to be able to utilize the total transmittance of the said lens system, the lens elements must in practice be coated with Anti Reflex coating film (AR-coating). If the said AR-coating films were not used, total transmittance would remain rather low.
In the HTS method, the use of fluorescent label molecules is restricted by the large number of samples; measurement results remain unreliable when using fluorometers because with large numbers of samples the output of the excitation lamps decreases, resulting in the need to service instruments even during operation. If, however, a combination consisting of several flash lamps is used, the lamp unit will be sufficiently powerful and the intervals for changing the lamp bulbs will be sufficiently long.
The aim of the invention is to achieve a versatile and efficient imaging device for luminescence, prompt fluorescence, time-resolved fluorescence, and absorbancy, that is, as for transmittance and photometric measurements.
The aim is to carry out mainly the following types of measurements with the device relating to the invention:
1. Prompt fluorescence
2. Time-resolved fluorescence (TR fluorescence)
3. Fluorescence polarization
4. Luminescence, such as chemiluminescence and electroluminescence
5. Absorbancy
6. SPA (=Scintillation Proximity Assay)
The present imaging device provides a solution to the problem of how to realize a sufficiently powerful lamp unit which produces a sufficiently short light pulse. This type of unit comprising several flash lamps is the only possible lamp for many time-resolved fluorescence measurements. One example of these is homogeneous fluorescence assay LANCE, which is a trademark of Wallac Oy. No such powerful lamp is known with which measurement can take place within a sufficiently short measuring time. The measuring time should be short, e.g. 2 minutes, in order for the total measuring time in High Throughput Screening to be acceptable. The total number of samples in one HTS run may be, for example, 100 000 samples, that is, sample wells. Measuring such a large number of sample wells by traditional fluorometric means would take an extremely long time. The amount of hazardous waste produced would also be considerably higher. A combination of a powerful pulsed laser and a dye laser could in principle be used, but it is an unnecessarily complex and uneconomical device which is also difficult to service.
The intervals for changing the lamp bulbs of the light source become extremely long when a lamp combination consisting of flash lamps is used. This is of considerable importance in HTS runs where the aim is to minimize the number of stoppages. When using conventional continuous, that is, continuous wave lamp units, for example, mercury or xenon arc lamps or halogen lamps, the lamp changing intervals are without exception too short considering the demands of HTS screening. One of the few possible solutions when using continuous lamps would be that the lamp could be changed automatically, but it is rather difficult to arrange this to function reliably.
The device relating to the invention combines the absorption measurements of both the near ultraviolet region and of visible light, or, vice versa, transmittance measurements, that is, in practice photometric measurements: the absorption of near UV is required especially in the applicant""s LANCE measurements. Absorption measurements in the visible region are in the nature of standards in terms of measuring technique.
The difficulties in using both absorption modes, such as near UV and visible region light mainly concern optics. It is almost impossible, or extremely uneconomical, to construct a telecentric lens with such a high light-collecting efficiency that would transmit effectively the wavelengths of both near UV and visible region light. Typically, a lens system which collects light efficiently and has good resolution can be made either in the near UV region, or alternatively in the wavelengths of visible light. This means that typically only absorption imaging in the visible region or absorption in the near UV light region are possible. In the application relating to the invention, on the other hand, a special scattering plate is used by means of which both near UV and visible light absorption measurements can be imaged.
The light source solution relating to the invention is advantageous for measuring numerous samples done by imaging, for example, especially in demanding applications such as HTS screening and both time-resolved and prompt fluorescence measurements. The device is able to image absorption measurements efficiently both in the near UV light region, that is, in 300-400 nm, and in the visible region, that is, 400-800 nm.
The size of a commonly used sample plate is 120xc3x9780 mm, which is imaged at one time, and a digital image is produced from it. In the example device, a sample area as large as 240xc3x97160 mm can be imaged in four parts one after another. The four images obtained in this way are combined into one by means of software. The sample may otherwise be of any shape, so long as it meets the boundary dimensions of the example situation. The amount of light produced by the sample and the desired resolution of details are decisive as regards the imaging efficiency of the device. The thickness chosen for the sample in this case equals plate thicknesses within the range of 0 . . . 30 mm.
In the device relating to the invention there are the following possible ways of illuminating or exciting the sample:
1. The sample can be illuminated and/or excited from above and/or below.
2. The sample can be imaged from above or below.
For measuring (imaging) the sample plate from below, the device is turned upside down.
Properties of the illumination system relating to the invention:
1. High average total pulse power
2. Narrow pulse width, which is required in the embodiment used as an example
3. The use of several lamps makes it possible to select the pulse energy of a single lamp so that the fluorescent sample will not become saturated. If a single lamp with high pulse power were used, the sample might become saturated, which would mean a sharp drop in the sensitivity of detection.
4. The use of several lamps also means that even if there should occur a fault in a single lamp, the overall efficiency remains almost unchanged in proportion to the number of lamps.
5. Using several pulse mode lamps also means that the intervals for changing the lamps are extremely long, for example, about six months. This is due to the fact that a pulse mode lamp is switched on only when it is really used. There is no wastage.
If, for example, a continuous arc lamp is used instead, the lamp will have to be changed at least at one month intervals. This is due to the fact that a continuous lamp is switched on all the time, that is, also when there is no sample in the sample space. It is not worthwhile, nor possible, to switch a continuous lamp off for example for a 15 minute waiting period, because the lamp reignites slowly due to the increase in the internal gas pressure inside the lamp, which is due to the warming up of the lamp following its use, this making it difficult, or even impossible, to reignite the lamp . In addition, stabilizing the arc lamp, that is, stabilizing its power within a desired wavelength region cannot be performed successfully if the lamp is switched on and off, for example, at 15 minute intervals.
A powerful light source is required in order for fluorescence measurement to be efficient. According to one embodiment, sufficiently strong excitation light is produced by using several lamps for the illumination, which can be switched on either simultaneously or successively by phasing.
If the lamps are switched on simultaneously, the simplest way is to position all the lamps in such a way that they can be directed towards the centre of the sample plate, and arrange the travel of the light beams by means of lenses so that the illuminating beams emitted from the lamps are distributed evenly across the entire sample plate from each lamp separately, but at the same time.
According to one embodiment, the lamps are situated on the circumference of a circle and their light is directed towards the centre of the circle, where there is a polygon mirror. In the polygon, there is an individual reflection plane for each lamp, the said plane reflecting the light beams of the lamp towards the centre of the sample plate. By means of the lenses, the light is distributed evenly over the entire sample plate area.
If the lamps are switched on successively by phasing, according to one embodiment the lamps are also situated on the circumference of a circle and their light is directed towards a rotating, for example, ellipse-shaped mirror in the centre of the circle. The rotating mirror is in an inclined position, for example, at a 45 degree angle, so that when the mirror rotates, it reflects the light from each flash lamp in turn onto the sample plate, preferably along the same optical path. This is implemented by triggering each flash lamp at the precise moment when the rotating mirror is in such a position during its movement that the illuminating beam emitted from the flash lamp at the moment of triggering strikes the reflecting surface of the rotating mirror so that the light beams are directed further towards the sample plate. The speed of rotation of the rotating mirror should not be too high, so that the random time lag resulting from the minor natural inaccuracy of the triggering moment between the triggering event and the flash of light from the lamp will not hinder the travel of the light beams in the desired optical path towards the sample plate. While the mirror is still rotating towards the next flash lamp, this lamp is being triggered in the same manner as the previous lamp and the light beams from this next lamp are directed in the same manner as above towards the sample plate. This function takes place at each individual flash lamp while the mirror is still rotating, with the result that during one full rotation of the rotating mirror, each flash lamp is triggered once, that is, each of the lamps has flashed once at precisely the appropriate moment, which corresponds to that point on the rotational angle of the mirror at each individual lamp, where the light beams are directed towards the sample plate.
The function described above only succeeds when pulse mode light sources are used which can be triggered. In such a case each flash lamp is controlled at full power which means for each individual lamp that the electric pulse fed to each individual lamp is exactly as high as the frequency complying with the rotation of the rotating mirror, that is, as high as the frequency, or cycle time, allows for each individual lamp. For example, if the speed of rotation of the rotating mirror is 6000 rpm (roots per minute), that is, one rotation lasts for 10 milliseconds, and if a lamp unit using, for example, eight flash lamps is used as a light source, in that case each of the eight lamps is triggered at 10 millisecond intervals, each at the precise moment when the rotating mirror is at the individual lamp in question. Since each individual lamp is triggered at 10 ms intervals, this means that each lamp is triggered 100 times per second, that is, at a frequency of 100 Hz. In the device application relating to the example a flash lamp with a maximum permitted average power of 50 watts (W) is used. In such a case, when the lamp is used at a frequency of 100 Hz, this means that 0.5 joules of energy is supplied to the lamp at each triggering. The said amount of energy is thus supplied to each lamp separately at the precise moment which corresponds to that point on the rotational angle of the rotating mirror at which it is at the individual lamp. Thus, in this example, each individual lamp is supplied with 100 Hz, that is, at intervals of 10 milliseconds, 0.5 joules energy is supplied to each lamp, which corresponds to an average power of 50 watts per each lamp separately. From this follows that the overall output of the lamp system as a whole is on average eight times 50 watts, that is, 400 watts. The overall output is, therefore, directly dependent on the total number of lamps.
In practice this means that each lamp is controlled, or triggered, with the highest average power permitted, that is, with 50 W. Due to the movement of the rotating mirror, each of the eight lamps in the lamp unit can be used at the full power permitted. In this way an average power output of 400 watts is produced by the system of eight lamps relating to the example.
The length of the pulse of a flash lamp is mainly only about 1 microsecond. However, the light pulse has a xe2x80x9ctailxe2x80x9d extending up to about 50 microseconds, the said tail limiting the sensitivity of measurements when the aim is to achieve extreme sensitivity. The operation of the flash lamp can be made more efficient by incorporating a so-called xe2x80x9ctail light chopperxe2x80x9d in the lamp unit. In that case each excitation light pulse should be shortened by positioning a device which functions like a fast shutter in front of the lamp, in order to cut off the excitation illumination path after each excitation light pulse. Thus, if the operation of this type of chopper were rapid enough, the light tail of the lamp could be cut, for example, 3 xcexcs after the moment of triggering the lamp, and in this way the flash lamp would produce an excitation light pulse lasting at longest 3 ps in total, which would extend the scope of application of the said lamp.
When measuring the fluorescent homogeneous sample referred to in the invention, it is sufficient if, for example, the light tail of the lamp is cut off about 30 xcexcs from the moment of triggering the lamp.
The tail light chopper can be implemented in several ways. Its operation can be combined with the operation of the rotating mirror, in such a way that if the rotating mirror rotates fast enough, due to this movement of the mirror, the tail of the light pulse is cut off timewise as the rotating mirror rotates further towards the location of the next lamp. In time-resolved measurements it is also important that enough time remains after excitation for measuring the emission signal before the next excitation pulse is directed at the sample. The rotating mirror can be rotated faster, in which case the cutting of the light tail becomes more accurate. In such a case it may, however, be more advantageous if the next lamp being triggered is the one after the next (in other words, every other lamp in the lamp system is triggered), in order to utilize the emission light of the fluorescence in an efficient manner. In this case there should preferably be an odd number of lamps in the lamp system, so as to make use of all the lamps. If there is an even number of lamps in the unit and the type of lamp triggering method described is to be used, care should be taken that the even number of lamps to be triggered is alternated at regular intervals. The selection can be automated by means of an electric coupling. This means that in a system comprising eight lamps, four lamps (such as, for example, lamps 1, 3, 5 and 7) are triggered first, and then after a regular interval, the remaining four lamps (that is, lamps 2, 4, 6 and 8 would be due to be triggered). When the group of lamps to be triggered is changed at regular intervals, all the lamps are used up evenly.
The structure of the lamp unit is not limited by the requirement of having an even or odd number of lamps.
The lamps in the lamp unit can also be triggered in other ways. The order of triggering can in itself be selected freely, because the selection is done by means of an electric circuit coupling or the program command of a computer processor, or a combination of these, and because this may give the advantage that the method of use, or the point of operation of an individual lamp can be made more appropriate considering, for example, the lamp""s service life or its UV light production, than by triggering the said lamps in strict order as described above.
Structurally, the tail light chopper may also be a separate mechanical light chopper which is located after the lamp so that the length of the aperture or the lengths and shapes of the apertures in it determine the true length of the light pulse. This type of tail light chopper is timed, that is, synchronized with an emission light chopper and the rotating mirror situated in the lamp unit, so that these operate together in a synchronous manner.
It is not worth using the rotating mirror system if continuous light sources are used as lamps. This is simply due to the fact that when the mirror rotates, and when each lamp is switched on all the time (because in such a case it would be a question of using continuous wave lamps, that is, continuous lamps), every time that the mirror is not exactly at the individual lamp, the optical power will be directed past the mirror most of the time, and thus not at the sample. On the other hand, if a fixed mirror polyhedron was to be used instead of the rotating mirror, in such a case both pulse mode and continuous light sources could be used. In that case the overall service life of the continuous light sources would naturally also depend on the number of lamps in the system, because whenever the service life of one continuous lamp came to an end, a new lamp would be switched on and operation could continue uninterrupted while the lamp was being changed.
The mechanical light chopper in front of the camera and on the emission light path must be synchronized with the movement of the rotating mirror in the lamp unit. The rotating mirror and the emission light chopper will then always at a certain phase rotate with respect to each other. Phase locking is performed electronically by means of the synchronizing pulses obtained from the emission light chopper and the rotating mirror. The synchronizing pulses are obtained from opto-electronic readers, or so-called N-coders, which rotate with the emission light chopper and the rotating mirror. If a tail light chopper is in addition used in front of the lamp, this also gives synchronizing pulses according to which the electronic unit guides the operation of the rotating means in a controlled manner.
In the solution described, the sensitivity of luminescence has been maximized, taking into account the requirements of structural simplicity. The result is affected by the structures of the large and small telecentric lenses, their coatings and the properties of the camera. For the duration of luminescence measurement, the mirrors in the mirror unit 22 are moved away so as not to obstruct the illumination path leading to the detector 11, and the filter wheel containing filters which is in the filter unit 12 is moved into such a position that there are either no filters in it or else there is a special luminescence filter. The rotating discs of the light chopper 13 are moved by means of the motors rotating the discs and the electric circuit coupling controlling the motors, into such a position that the illumination path leading to the detector 11 is unobstructed.